| Hepatic ischemia-reperfusion injury is attracting great attention in clinic. It is common in many pathologic processes and surgeries,such as hemorrhagic shock, hepatectomy and liver transplantation. Especially,in liver transplan tation,it is unavoidable and has no ideal method for prevention and treatment. With the advances in research of MSCs, the multipotential differentiation of it has been proved. It can not only differentiate into interstitial cells of mesoderm, but also the cells of other germinal layers, such as nerve cells and hepatic cells. It was found in vivo that MSCs could transfer, locate and differentiate into corresponding tissue and cells, participating in the repair of injured tissue and organs. The effect and mechanism of bone marrow mesenchymal stem cells in repairing the hepatic ischemia-reperfusion injury were studied.Objective:To study the method of isolated culture of bone marrow mesenchymal stem cells (MSCs), to establish the rat model of hepatic ischemia-reperfusion, and observe the repairative effect and mechanism of MSCs in the hepatic ischemia-reperfusion injury.Methods:The cells were expanded by subculture and the threeth passage cell, proven to be capable of differentiation into osteoblasts and lipocyte, were injected into the injured model rats via. Rat bone marrow mesenchymal stem cells were cultured in vitro. Rats were divided randomly into the treated group and the negative control group. The treated group were injected MSCs 0.5mL, the control group were injected normal saline 0.5mL by tail vein. The normal rats were blank control group. We used injured hepatic tissue to induce the differentiation of MSCs .The specific markers of hepatic cell, such as albumin, were detected by immunocytochemical staining. Then we observed the site of DAPI fluorescent labeling MSCs under laser confocal microscopy. Y bodies were detected by agarose gel electrophoresis to confirm donorcells homing receptor tissue. Morphologic changes and cell apoptosis status of liver would be observed under light microscopy. The mice in experimental group, negative control group and blank control group were killed after one, two and four weeks, respectively. AST, ALT, GT, AKP, TP, ALB, A/G, GSH-Px, GSH, GR in serum and SOD, MDA, TP, and ALB in tissue were detected and analized respectively.Results:1. MSCs in high purity and with character of adherence were obtained. MSCs submit hypodispersion, colony-like growth and fusiform shape in primary culture. MSCs obviously improve homogeneity in serial subcultivation and passage is very stable.2. Immunocytochemical staining: We can detect the expression of ALB in group of injured hepatic tissue induced differentiated MSCs after 21 days. The cells in control without induce were negative. MSCs were induced into hepatocyte-like cells in vitro. 3. Laser confocal microscopy:Fluorescence was detected in injured hepatic tissue 3 days later after DAPI fluorescent labeling. MSCs with DAPI fluorescent labeling arrived in the area of injured hepatic tissue. 4. Agarose gel electrophoresis: fragments about 100bp were amplified. Y bodies were detected. MSCs could transfer, locate, survive, and differentiate into required cells, in order to promote the repair of injured tissue and organs.5. Morphologic changes of liver: no obvious necrosis was found in experimental group 2 weeks later after transplantation. And the hepatic cord was repaired, with some little round lipid droplet. Obvious necrosis was found in controls. 4 weeks later, the structure of hepatic tissue tended to be normal in experiment group, with infiltration of few inflammatory cells; while fibrosis were found in controls.6. The levels of AST, ALT in experiment group decreased continually, which showed the liver function had been much improved. ALT showed different in 2 weeks and 4 weeks compared to control; AST showed in 4 weeks. The level of SOD in the experiment group increased, and MDA decreased compared to control, which showed different 2 weeks and 4 weeks later after injection into cells.7. Bax, Bcl-2, Caspase-3 increased obviously 1 weeks after injection of MSCs. The expression of positive cells decreased obviously from 2 weeks later. The ratio of Bcl-2 and Bax became increased from 2 weeks later, and went to the peak 4 weeks later which is nearly same with the ratio in 2 weeks. Apoptosis had been controlled from 2 weeks later.Conclusions:1. A method for isolation and purification in vitro of MSCs from bone marrow has been established. It is observed by morphous and bionomics. It suggests that the method of isolation, high purification and active proliferation of MSCs by adherent culture and tissue blot culture in special culture system is very convenient and effective. 2. MSCs transfusion by caudal vein can repair the ischemia-reperfusion injury of liver, is helpful to the structure and function recovery of injured liver. 3. There are many possible mechanisms of repairment of MSCs to the ischemia- reperfusion injury of liver. The directional migration of MSCs and differentiate into hepatocyte-like cells is important one of them.
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